Microfluidics: an enabling technology for the life sciences

During the last year we have investigated existing and future markets, products and technologies for microfluidics in the life sciences. Within this paper we present some of the findings and discuss a major trend identified within this project: the development of microfluidic platforms for flexible design of application specific integrated microfluidic systems. We discuss two platforms in detail which are currently under development in our lab: microfluidics on a rotating CD ("Lab-CD") as well as a platform to realized customized "nanoliter & picoliter dispensing systems"

Schizophrenic molecules and materials with multiple personalities - how materials science could revolutionise how we do chemical sensing

Molecular photoswitches like spiropyrans derivatives offer exciting possibilities for the development of analytical platforms incorporating photo-responsive materials for functions such as light-activated guest uptake and release and optical reporting on status (passive form, free active form, guest bound to active form). In particular, these switchable materials hold tremendous promise for microflow-systems, in view of the fact that their behaviour can be controlled and interrogated remotely using light from LEDs, without the need for direct physical contact. We demonstrate the immobilisation of these materials on microbeads which can be incorporated into a microflow system to facilitate photoswitchable guest uptake and release. We also introduce novel hybrid materials based on spiropyrans derivatives grafted onto a polymer backbone which, in the presence of an ionic liquid, produces a gel-like material capable of significant photoactuation behaviour. We demonstrate how this material can be incorporated into microfluidic platforms to produce valve-like structures capable of controlling liquid movement using light

Vortex fluidics-mediated DNA rescue from formalin-fixed museum specimens.

DNA from formalin-preserved tissue could unlock a vast repository of genetic information stored in museums worldwide. However, formaldehyde crosslinks proteins and DNA, and prevents ready amplification and DNA sequencing. Formaldehyde acylation also fragments the DNA. Treatment with proteinase K proteolyzes crosslinked proteins to rescue the DNA, though the process is quite slow. To reduce processing time and improve rescue efficiency, we applied the mechanical energy of a vortex fluidic device (VFD) to drive the catalytic activity of proteinase K and recover DNA from American lobster tissue (Homarus americanus) fixed in 3.7% formalin for >1-year. A scan of VFD rotational speeds identified the optimal rotational speed for recovery of PCR-amplifiable DNA and while 500+ base pairs were sequenced, shorter read lengths were more consistently obtained. This VFD-based method also effectively recovered DNA from formalin-preserved samples. The results provide a roadmap for exploring DNA from millions of historical and even extinct species

Developing Droplet Based 3D Cell Culture Methods to Enable Investigations of the Chemical Tumor Microenvironment

Adaptation of cancer cells to changes in the biochemical microenvironment in an expanding tumor mass is a crucial aspect of malignant progression, tumor metabolism, and drug efficacy. In vitro, it is challenging to mimic the evolution of biochemical gradients and the cellular heterogeneity that characterizes cancer tissues found in vivo. It is well accepted that more realistic and controllable in vitro 3D model systems are required to improve the overall cancer research paradigm and thus improve on the translation of results, but multidisciplinary approaches are needed for these advances. This work develops such approaches and demonstrates that new droplet-based cell-encapsulation techniques have the ability to encapsulate cancer cells in droplets for standardized and more realistic 3D cell culture and cancer biology applications. Three individual droplet generating platforms have been designed and optimized for droplet-based cell encapsulation. Each has its own advancements and challenges. Together, however, these technologies accomplish medium to high-throughput generation (10 droplets/second to 25,000 droplets/second) of biomaterial droplets for encapsulation of a range of cell occupancies (5 cells/droplet to 400 cells/droplet). The data presented also demonstrates the controlled generation of cell-sized small droplets for biomolecule compartmentalization, droplets with diameters ranging between 100-400 μm depending on device parameters, and the generation of instant spheroids. Standardized assays for analyzing cells grown within these new 3D environments include proliferation assays of cells grown in mono- and co-cultures, the generation of large and uniform populations of scaffold supported multicellular spheroids, and a new system for culturing encapsulated cells in altered environmental conditions

Microfluidic cell sorting techniques to study disease processes.

Circulating nucleated cell populations found in whole blood, including both white blood cells (leukocytes) and endothelial cells, provide an ideal platform for studies seeking to understand the disease processes for development of drugs and treatments. This thesis presents an automated microfluidic device developed for leukocyte enrichment from peripheral blood. Briefly, the device allows for complete lysis of red blood cells and comprehensive analysis of nucleated cell populations in terms of quantity and activation status. The microfluidic lysis device was used in two Sickle Cell disease (SCD) studies to understand the effect of leukocytes in the initiation of vasoocclusive crisis. Findings suggest abnormally high baseline leukocyte counts and variance in clinical expression among SCD patients. Hence, a highly favorable state for an inflammatory reaction that may lead to vasoocclusive episodes exists. To ascertain risk factors in such incidents revision of current SCD patient classification is needed

Interfacing to Biological Systems Using Microfluidics

Biological systems operate on scales ranging from nanoscale chemical reactions to the global flow of nutrients and energy. Building knowledge of each level requires techniques and technologies that can address the biological system at the chosen level of interest. On the cellular and community levels, microfluidics are able to replicate the spatial scales of the natural system from the cellular, to community through the local microenvironment while providing engineering solutions to control flow through the system and interfaces with the system through microscopy and chemical sampling. Herein, biological interfaces were created using microfluidics to control cellular interactions and chemical reactions. At the subcellular scale, molecular exchange bioreactors enhanced the protein production of a cell-free protein synthesis system by using a microscale serpentine channel to reduce lateral diffusion distances. Size dependent transport of reactants into, and byproducts out of, the reaction channel through the nanoporous barrier extended the reaction time and enhanced protein yield. Nanoporous membranes were also developed for studying cellular interactions. Membranes confined cells within culture chambers while allowing transport of nutrients and signal molecules between the chambers and support channels. Quorum sensing within the microfluidic chambers was modeled using a quasi-steady-state PDE based approach to estimate relative concentrations. The platform facilitated the use of brightfield imaging and analysis to characterize morphological changes of a growing biofilm as the oral microbe Streptococcus gordonii formed aggregates only when co-cultured adjacent to Fusobacterium nucleatum. The investment of capital and time to start incorporating microfluidic into research can be prohibitive. To combat this, tools were created to provide researchers the ability to create microfluidics using 3D printing to simplify the process and remove the need for cumbersome and expensive cleanroom facilities. The technique was used in two common microfluidic applications of chemical gradient and droplet formation in addition to building 3D fluidics that cannot be replicated directly with microfabrication techniques. These microfluidics controlled the spatiotemporal environment on the scales of biological systems to enhance the effectiveness of protein synthesis, give insight to morphological effects of cell signaling, and introduced technology to enable others to do the same

Physics and Applications of a PDMS Based Centrifugal Microfluidic System

The objective of this research work is to develop a centrifugal microfluidic system for general purposes based on microfabrication technologies including SU-8 photolithography, polydimethylsiloxane (PDMS) casting. The main contribution of this research is to integrate a flyball governor system into the polymer based centrifugal microfluidic platform. A series of function units are developed based on this unique mechanism. In the first part, three pinch valve systems were designed and tested. The first one is based on the magnetic force and the second one is on the basis of spring force and the last one is a membrane valve. All valving system demonstrate good control of the fluid movement. The latter two valves are capable of sequential control. It proves that the flyball governor system is very compatible with centrifugal fluidic technologies. The major advantage of this new actuation technology is that its burst frequency can be conveniently manipulated by adjusting the parameters of the mechanical system without changes in the fluidic pattern. Next, two types of inward pumping systems were designed and tested. The result shows that both the inward pumps were capable of the pumping over a radial distance of 21mm in a short time. It thus improves the usage of space on the disc and paves the way to interconnect several functional units. Then as a proof of concept, a sequential valving system capable of metering and centrifugal sediment was developed for plasma extraction from whole blood. The resulting residual cell concentration was less than 0.5%. In the last part, a micromixer was developed based on the similar principle. The results show that the flyball governor system can effectively agitate the chaotic mixing of the sample liquids by periodically deflecting the PDMS membrane of the mixing chamber. The mixing effect can thus be enhanced

A microfluidic culture for two populations of dorsal root ganglia for differential staining

The goal of this study was to design and fabricate a microfluidic system that can be used to visibly distinguish the two populations of dorsal root ganglia (DRGs) by differential staining. Polydimethylsiloxane (PDMS) is the most widely used silicon-based organic polymer, and is particularly known for its wide spread use in microfluidics. Various methods have been employed to pump fluids in these channels for applications ranging from patterning of cells and biomolecules to control of local environment factors such as temperature, which requires external pumping or other applied forces. We demonstrated a pump-free device that exploits the surface energy stored in a liquid droplet to pump liquid in the channels. The fluid was pumped by using two droplets of unequal sizes connected via fluid filled channel. The flow was generated from smaller droplet to larger droplet. This passive pumping technique was used to simultaneously stain the two cultured DRGs in connected channels. The in vitro system can be further exploited to study the guided growth in axons. This study provides a cost effective method to detect the influence of the presence of pioneer neuron on the growth patterns of the new generation of neurons. It eliminates the need of using transgenic cells to study the guided growth in axons, thereby giving some insight for the repair of spinal cord injuries and the understanding of the early growth model